Cell-free synthesis of proteins with selectively ¹³C-labelled methyl groups from inexpensive precursors

The novel eCell system maintains the activity of the entire repertoire of metabolic Escherichia coli enzymes in cell-free protein synthesis. We show that this can be harnessed to produce proteins with selectively ¹³C-labelled amino acids from inexpensive ¹³C-labelled precursors. The system is demonstrated with selective ¹³C labelling of methyl groups in the proteins ubiquitin and peptidyl-prolyl cis-trans isomerase B. Starting from 3-¹³C-pyruvate, ¹³C-HSQC cross-peaks are obtained devoid of one-bond ¹³C-¹³C scalar couplings. Starting from 2-¹³C-methyl-acetolactate, single methyl groups of valine and leucine are labelled. Labelling efficiencies are 70 % or higher, and the method allows us to produce perdeuterated proteins with protonated methyl groups in a residue-selective manner. The system uses the isotope-labelled precursors sparingly and is readily scalable.